腐食酪螨过敏原Tyr p 5生物信息学分析及原核表达

Characterization and prokaryotic expression of group 5 allergens from Tyroph-agus putrescentiae

目的:克隆表达腐食酪螨过敏原第5组分(Tyr p 5),并了解其分子特征。方法:采用TaKaRa MiniBEST Universal RNA Extraction Kit试剂盒提取腐食酪螨总RNA,根据GenBank已公布的Tyr p 5核酸序列设计引物,用RT-PCR合成Tyr p 5编码基因并与pET-28a(+)载体连接后转入大肠杆菌E.coli BL21诱导表达,通过Ni柱亲和层析纯化得到蛋白。运用生物信息学方法预测分析Tyr p 5蛋白的结构和功能。结果:从腐食酪螨cDNA中扩增获得Tyr p 5基因片段,成功构建了表达质粒pET-28a(+)-Tyr p 5,Western blot结果显示原核表达获得成功。获得的Tyr p 5基因与参考序列同源性(GenBank AY800360)达99.8%,含有一个完整的开放阅读框,由158个氨基酸组成,信号肽位于1~36AA、跨膜区域为23~33AA,为细胞外亲水蛋白。Tyr p 5蛋白的二级结构由75.32%的α螺旋、1.9%的β转角和22.78%的无规则卷曲构成。α螺旋为Tyr p 5蛋白的主要折叠形式。其氨基酸序列与棉兰皱皮螨过敏原第5组分相似率为52.68%,分子进化树中腐食酪螨与棉兰皱皮螨聚成一簇,并与现行的形态学分类系统一致符合。结论:成功克隆、表达、纯化腐食酪螨Tyr p 5蛋白,并初步预测其分子特征。

Objective:To clone and express Tyr p 5 protein and predict its molecular characteristics.Methods:The total RNA of T.putrescentiae were extracted by using TaKaRa MiniBEST Universal RNA Extraction Kit,and the Tyr p 5 gene was amplified by RT-PCR according to previous sequence published in GenBank,and then connected with pET-28a(+)vector and then transferred to Escherichia coli.The expression of E.coli BL21 was induced,and the protein was purified by Ni column affinity chromatography.The structure and function of the Tyr p 5 protein was analyzed by using bioinformatics software.Results:The Tyr p 5 gene fragment was amplified from the cDNA of Tyr P 5,and the expression plasmid pET-28a(+)-Tyr p 5 was successfully constructed.Western blot analysis showed prokaryotic expression achieved success.The sequence homology reached to 99.8%between our sequenced result with one complete open reading fragment(ORF)and the reference(GenBank AY800360).The gene encode an extracellular hydrophobic protein with 158 amino acid resides,one signal peptide from l to 36 position and one transmembrane domain from 23 to 33 position.Secondary structure analysis revealed that Tyr p 5 contained anαhelix(75.32%),aβturn(1.9%),and a random coil(22.78%).Theαhelix was the major folding form of Tyr p 5 protein.The similarity of the amino acid sequence of the group 5 allergens were 52.68%between T.putrescentiae and S.medanensis,Which was consistent with morphologic taxonomy adopted by most of acarologists.Conclusion:The allergen Tyr p 5 with strong allergenicity was cloned,expressed and purified,and its molecular characteristics was preliminarily predicted.